Toxoplasma gondii genotyping in AIDS patients using high resolution melt analysis (HRM analysis) in Khuzestan province, southwest Iran.

Toxoplasmosis is a parasitic zoonotic disease caused by Toxoplasma gondii, a common protozoan in the Apicomplexa phylum. Several studies in Iran have demonstrated the presence of the parasite in various hosts, but no data on T. gondii genotyping in HIV patients in Khuzestan, Southwest Iran, is available. One hundred of blood samples from AIDS patients were collected and tested by real-time PCR High Resolution Melting analyses for T.gondii detection and genotyping. T. gondii was discovered in 8 out of 100 (8%) AIDS patients with dominant Type I. This study suggest that HRM method demonstrated excellent discriminating ability for T. gondii, and AIDS patients should be tested for Toxoplasma detection and genotyping to prevent parasite pathogenicity.

Seroprevalence and molecular detection of Toxoplasma gondii in healthy blood donors in southwest Iran.

BACKGROUND: Toxoplasmosis is a cosmopolitan parasitic disease caused by Toxoplasma gondii (T. gondii). Blood transfusion is a probable route of T. gondii transmission. Due to lack of information about seroprevalence of T. gondii in healthy blood donors, this study was aimed to determine the chronic and acute infection using serological and molecular methods. MATERIAL AND METHODS: In this cross-sectional investigation, 380 samples were collected from donated bloods. Anti-Toxoplasma IgG and IgM antibodies were examined using enzyme-linked immunosorbent assay (ELISA). Also, all IgG positive samples were tested by IgG avidity test. Eventually, to detection of active infection, DNA was extracted from IgM positive and low IgG avidity samples and then tested using nested-polymerase chain reaction (PCR). RESULTS: Among 380 blood donors, 131 (34.47%) were positive for only anti-T. gondii IgG, 2 (0.5%) were positive for only anti-T. gondii IgM, and 11 (2.9%) were positive for both IgG and IgM antibodies. Then, 142 samples (131 IgG + and 11 IgG +IgM +) were evaluated using IgG avidity test. Of these, 115 (81%) had high avidity IgG indicates past infection; 16 (11.26%) had low avidity IgG representing recent infection, and 11 (7.74%) were equivocal. With nested PCR, 20 samples of 50 seropositive samples were diagnosed positive. CONCLUSION: Detected active infection using nested-PCR draws attention to the possibility of T. gondii infection via blood transfusion which emphasizes the importance of parasite DNA screening before donation of blood in high risk groups such as: multi-transfused persons, immunosuppressed patient, and pregnant women.

Detection and genotyping of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran.

To detection and genotype of Toxoplasma gondii isolated from soil in Ahvaz, southwest of Iran. Between August 2011 and May 2012 at different sites located in the area of the Ahvaz city south west Iran. A total of 200 soil samples were taken from different points of the region. Oocysts were recovered using the flotation method. Then, PCR reactions targeting the GRA6 gene were performed for specific T. gondii detection. The positive samples were studied by RFLP (random amplified fragment length polymorphism) using MseI enzymes to confirm the parasite linage. Toxoplasma DNA was found in 18 samples. Among them, 12 samples were successfully genotyped as GRA6 type III and 6 as GRA6 Type II. This is the first investigation detecting and genotyping T. gondii oocyst in environmental soil samples of Ahvaz, South west of Iran. The results of this study indicated that soil contaminated with T. gondii oocysts especially in public park may play a role in the epidemiology of human toxoplasmosis in southwest of Iran.

Seroprevalence of Toxoplasma gondii in women who have aborted in comparison with the women with normal delivery in Ahvaz, southwest of Iran.

Toxoplasma gondii is an obligate intracellular protozoan parasite causing toxoplasmosis in animals and humans. Primary maternal infection with toxoplasmosis during pregnancy is frequently associated with transplacental transmission to the fetus. However it is not certain whether Toxoplasma infection can cause recurrent abortion. The aim of this study was to determine the relationship between Toxoplasma infection and abortion via detection of anti-Toxoplasma gondii antibodies in sera of women with obstetrical problems and compare the results with control group consisting of women with history of normal delivery. Sera from 130 women with abortion and sera of 130 women with normal delivery were tested for IgG and IgM anti-Toxoplasma gondii antibodies by ELISA method. The present study revealed 24.6% of the samples with abortion and 21.5% of the samples with normal delivery were positive for IgG antibodies. However, statistical analysis indicated no significant differences (P > 0.05). In addition, IgM antibody was detected in one woman who had aborted but not in women with normal childbirth. This study showed no significant difference between the case and control groups in IgG anti-Toxoplasma antibody but detected one sample with IgM antibodies in woman with abortion during the first trimester of pregnancy. In order to determine the relationship between Toxoplasma infection and abortion, anti-Toxoplasma IgG avidity and PCR to discriminate between recent and prior infections are recommended.

Detection of Toxoplasma gondii by PCR and mouse bioassay in rodents of Ahvaz district, southwestern Iran.

Toxoplasma gondii is obligate coccidian zoonotic parasite. Felidae family is definitive and wide ranges of warm-blooded vertebrates are intermediate hosts for the parasite. Rodents are measured as an important source of T. gondii infection for the definitive host. Thus, this study aimed to investigate Toxoplasm infection in rodents of Ahvaz district, southwest of Iran. A total of 100 rodents (73 Rattus norvegicus, 21 Rattus rattus, and 6 Mus musculus) were collected and studied by GRA6PCR and mouse bioassay. The finding indicated that 6 out of 100 (6%) and 2 out of 100 (2%) samples were positive by PCR and mouse bioassay, respectively. The results show notable chronic infection in the rodent and potential transmission of the infection among animal and men in the region. Accordingly, this study recommended investigating of the T. gondii infection in definitive and other intermediate hosts in other points of Khuzestan province, Southwest, Iran.

Detection and genotyping of Toxoplasma gondii strains isolated from birds in the southwest of Iran.

1. The aim of this work was to determine the frequency of occurrence of Toxoplasma gondii and genetically analyse isolates from a number of avian hosts in the southwest of Iran (Khuzestan province). The frequency of T. gondii was determined in free-range chickens (Gallus domesticus), sparrows (Passer domesticus), pigeons (Columba livia) and starlings (Sturnus vulgaris). 2. Isolates obtained from Toxoplasma-infected birds were subjected to molecular typing by PCR-restriction fragment length polymorphism (RFLP) with sequence analysis of the GRA6 gene. 3. The results showed that 41 (16·5%) of 241 samples of avian tissue were infected with T. gondii. Sparrows were most frequently infected (17 out of 64). 4. Analysis of the GRA6 gene by PCR-RFLP and DNA sequencing revealed Type II and III T. gondii were the predominant lineage, accounting for 19·5% and 80·5% of the isolates, respectively. 5. It was concluded that the use of this PCR test facilitated the diagnosis of T. gondii in avian hosts and the GRA6 PCR-RFLP method clearly differentiated between the three different T. gondii lineages. This study showed a higher prevalence of type III compared with type II T. gondii in infected avian hosts in southwestern Iran.

Molecular characterization of livestock and human isolates of Echinococcus granulosus from south-west Iran.

Hydatidosis is an important public health problem in several parts of Iran. The aim of this molecular study is to investigate Echinococcus granulosus genotypes as the causative agents of hydatidosis in the south-west of Iran (Khuzestan province). In this study, isolates of 334 hydatid cysts were collected from the liver and lungs of 141 sheep, 104 cattle, 84 goats and 5 human cases. DNA was extracted and examined by nested polymerase chain reaction (PCR) of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and restriction fragment length polymorphism (RFLP)-PCR. In addition, fragments of genes coding for ITS1 were sequenced. The results of RFLP-PCR analysis revealed the presence of the G1 genotype in all human, cattle, goat and sheep isolates. Furthermore, no camel strain (G6) was detected among all samples in the regions studied. The molecular findings indicate that the predominant genotype involved in E. granulosus transmission in south-west Iran is the common sheep strain (G1 genotype), which occurs in human, cattle, sheep and goat populations. In conclusion, these results may have important implications for hydatid disease control in the areas studied.

Detection of toxoplasmosis in patients with end-stage renal disease by enzyme-linked immunosorbent assay and polymerase chain reaction methods.

Toxoplasmosis caused by Toxoplasma gondii is an opportunistic infection. In healthy individuals, the infection is largely asymptomatic, but in immunocompromised people the parasite can become widely disseminated, causing severe toxoplasmosis. In patients undergoing haemodialysis, the phagocytic process shows a highly significant impairment. Therefore, this study aimed to investigate toxoplasmosis in patients with end-stage renal disease (ESRD) undergoing haemodialysis in Ahvaz hospitals, southwest of Iran. A total of 280 patients and 100 healthy subjects participated in this study. The presence of serum IgM and IgG antibodies against T. gondii was detected by ELISA and the presence of Toxoplasma parasites in whole blood was evaluated by GRA6 PCR. Anti-T. gondii IgG antibodies were detected in 82 (29.3 %) haemodialysis patients and 26 (26 %) controls. In addition, anti-T. gondii IgM antibodies were detected in 7.9 % of patients and in 4 % of controls. For both the antibodies, the differences were statistically significant (P < 0.05). PCR was performed with DNA extracted from blood samples of all patients and controls. PCR gave positive results with four of the 280 blood samples from patients but none for the control blood samples. The results revealed a high percentage of positivity for Toxoplasma antibodies in patients with ESRD undergoing haemodialysis and also confirmed the parasite in whole blood, indicating disseminated infection in these patients. Patients undergoing dialysis have a higher rate of active infection with Toxoplasma likely due to reactivation of a chronic infection. Thus, parasitological examinations of ESRD patients should be periodically carried out for monitoring and evaluating the possible dissemination of toxoplasmosis during haemodialysis.

Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

BACKGROUND: The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. MATERIALS AND METHODS: Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. RESULTS: One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. CONCLUSION: This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

Mini-exon genotyping of leishmania species in khuzestan province, southwest iran.

BACKGROUND: Leishmaniasis is a protozoan disease cause by Leishmania genus. Anthroponotic and zoonotic cutaneous leishmaniasis are endemic in Iran. The aim of this study was to identify the causative agent of cutaneous leishmaniasis by mini-exon gene in five regions of Khuzestan Province, southwest of Iran. METHODS: From 2007 to 2008 in this cross-sectional study, cutaneous samples were collected from patients referred to Health Centers and Hospitals of the Khuzestan Province for cutaneous leishmaniasis diagnosis and cultured in Novy-MacNeal-Nicolle (NNN) and RPMI 1640. The propagated promastigotes were harvested and Leishmania species of cutaneous leishmaniasis were identified by RFLP and DNA sequencing of the PCR generated fragments. RESULTS: L. major and L. tropica were the causative agents of cutaneous leishmaniasis by predominantly of L. major species. The alignment of the mini-exon sequencing isolates with reported sequencing of L. major and L. tropica revealed 92%-99% identity. CONCLUSION: Our study showed that mini-exon PCR-RFLP was useful method to identify the causative species of cutaneous leishmaniasis.